Flavonoids are a diverse group of secondary metabolites that are synthesized in plants and have various biological functions. They are involved in plant defense against insects, pathogens and microbes and in absorption of free radicals and UV light. They also can act as pigments that attract beneficial symbionts and pollinators. Because the flavonoids are important for optimal plant growth and thus maximal agricultural productivity, the biochemistry and molecular biology of flavonoids is an important and very advanced area of research. Much of the knowledge in this field was reviewed by (Saslowsky, D., and Winkel-Shirley, B. (2001). Localization of flavonoid enzymes in Arabidopsis roots. Plant J 27, 37-48.; Winkel-Shirley, B. (2002). Biosynthesis of flavonoids and effects of stress. Curr Opin Plant Biol 5, 218-223.; Dixon, R. A., Xie, D. Y., and Sharma, S. B. (2005). Proanthocyanidins—a final frontier in flavonoid research? New Phytol 165, 9-28; Lepiniec, L., Debeaujon, I., Routaboul, J. M., Baudry, A., Pourcel, L., Nesi, N., and Caboche, M. (2006). Genetics and biochemistry of seed flavonoids. Annu Rev Plant Biol 57, 405-430). FIG. 1 shows an outline of the flavonoid biosynthetic pathway and a summary of biological functions of a few key metabolites. Enzymes involved in the pathway are listed in a sequential order (top to bottom): PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; CHS, chalcone synthase; AS, aureusidin synthase; CHI, chalcone isomerase; FS1/FS2, flavone syntase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol-4-reductase; LDOX (ANS), leucoanthocyanidin dioxygenase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; OMT, O-methyltransferase; UFGT, UDP-glucose:flavonoid 3-O-glucosyltransferase; RT, rhamnosyl transferase; C/EC refers to catechins/epicatechins, PPO refers to polyphenol oxydase.
Uses
The cocoa tree, Theobroma cacao, normally produces small amounts of epicatechin oligomers, commonly termed proanthocyanidins. These epicatechin oligomers are highly desired as they are potent antioxidants and thereby possess valuable properties as antioxidants, anti-inflammatories, and antiviral and antibacterial and antiparasitic agents. They have also been implicated in inhibition of low-density lipoprotein oxidation, vasodialation and reduction of hypertension, inhibition of platelet activation, and thus have many potential medical applications Hannum, S. M., and Erdman, J. W. (2000). Emerging health benefits from cocoa and chocolate. J Med Food 3, 73-75; Keen, C. L., Holt, R. R., Polagruto, J. A., Wang, J. F., and Schmitz, H. H. (2002). Cocoa flavanols and cardiovascular health. Phytochem Rev 1, 231-240; Fisher, N. D., and Hollenberg, N. K. (2005). Flavanols for cardiovascular health: the science behind the sweetness. J Hypertens 23, 1453-1459; Engler, M. B., and Engler, M. M. (2006). The emerging role of flavonoid-rich cocoa and chocolate in cardiovascular health and disease. Nutr Res 64, 109-118; Norman, K. H., Naomi, D. L. F., and Marjorie, L. M. (2009). Flavanols, the Kuna, cocoa consumption, and nitric oxide. J. Am. Soc. Hypertens 3, 105-112).
Enzymology
All flavonoids are derived from cinnamic acid, a derivative of the amino acid phenylalanine. Their biosynthetic pathways share some general steps and most start from the condensation of three malonyl-CoA units and p-coumaroyl-CoA catalyzed by chalcone synthase (CHS) to produce tetrahydroxychalcone. Yellow-colored tetrahydroxychalcone is then converted into the colorless naringenin through the stereospecific isomerization by chalcone isomerase (CHI) Dixon, R. A., and Paiva, N. L. (1995). Stress-induced phenylpropanoid metabolism. Plant Cell 7, 1085-1097; Holton, T. A., and Cornish, E. C. (1995). Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7, 1071-1083). In legume species, tetrahydroxychalcone can also be reduced to trihydroxylchalcone by chalcone reductase (CHR), and then converted into liquiritigenin by CHI Welle, R., and Grisebach, H. (1989). Phytoalexin synthesis in soybean cells: elicitor induction of reductase involved in biosynthesis of 6′-deoxychalcone. Arch Biochem Biophys 272, 97-102).
Naringenin enters into different pathways as a substrate for the synthesis of six different groups of flavonoids. It can be converted into dihydroflavonols by flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H) or flavonoid 3′,5′-hydroxylase (F3′5′H). Dihydroflavonols can then be converted into flavonols by flavonol synthase (FLS) and anthocyanins by a series of enzymes including dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS), UDP-glucose flavonol 3-O-glucosyl transferase (UFGT). Alternatively, naringenin can be converted by isoflavone synthase into isoflavones, which are the precursor for the synthesis of isoflavonoids. Naringenin can also be converted by DFR into flavan-4-ols, which are the precursors of 3-deoxyanthocyanidins, or it can be converted into flavones by flavone synthase 1 and 2 (FS1/FS2). One set of intermediates in the anthocyanin synthesis pathway, leucoanthocyanidins and 3-OH-anthocyanins are converted into the flavan-3-ols (catechin and epicatechin), which are polymerized into proanthocyanidins (condensed tannins) that are the major topic of this application.
Formation and Structures of Proanthocyanidins
The synthesis of proanthocyanidins (PAs) and anthocyanins shares common steps in the flavonoid biosynthesis pathway up to the synthesis of flavan-3,4-diols (such as leucoanthocyanidin), which not only are precursors for anthocyanin and flavan-3-ols synthesis, but also contribute to the extension units of the PA polymers (FIG. 1-2)(Dixon, R. A., Xie, D. Y., and Sharma, S. B. (2005). Proanthocyanidins—a final frontier in flavonoid research? New Phytol 165, 9-28). Flavan-3-ols (sometimes referred to as flavanols, such as catechin or epicatechin) derived from leucoanthocyanidins are believed to act as terminal units to initiate PA polymerization, while intermediates derived from leucoanthocyanidins themselves act as extension units to add to flavan-3-ol initiators through C4-C8 linkage (dominant form of PAs or C4-C6 linkage to form branches (not shown).
The hydroxylation pattern of the B-ring of the monomeric proanthocyanidins is determined by the presence of the cytochrome P450 monooxygenases flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H), enzymes that act early in the flavonoid synthesis pathway after the formation of naringenin (Winkel-Shirley, B. (2002). Biosynthesis of flavonoids and effects of stress. Curr Opin Plant Biol 5, 218-223; Dixon, R. A., Xie, D. Y., and Sharma, S. B. (2005). Proanthocyanidins—a final frontier in flavonoid research? New Phytol 165, 9-28) (FIG. 1). In the absence of both of these cytochrome P450 enzymes, hydroxylation occurs only at the 4′ position of B rings, yielding (epi)afzelechin. In the presence of F3′H, the 3′ position will be hydroxylated resulting in the formation of (epi)catechin. In the presence of F3′5′H, the 5′ position will also be hydroxylated leading to the formation of (epi)gallocatechin. The activity of F3′H and F3′5′H will also cause similar hydroxylation pattern on the B-ring of anthocyanins, resulting in the formation of pelargonidin with only one hydroxyl group, cyanidin with two hydroxyl groups, and delphinidin with three hydroxyl groups. The pigments derived from each anthocyanin have a characteristic color range since the visible absorption maximum becomes longer with the increase in B-ring hydroxyl groups: pelargonidin derived pigments show orange, pink or red colors, cyanidin-derived pigments show red or magenta colors and delphindin-derived pigments show purple or blue colors (Zuker, A., Tzfira, T., Ben-Meir, H., Ovadis, M., Shklarman, E., Itzhaki, H., Forkmann, G., Martens, S., Neta-Sharir, I., Weiss, D., and Vainstein, A. (2002). Modification of flower color and fragrance by antisense suppression of the flavanone 3-hydroxylase gene. Mol. Breed. 9, 33-41).